Optimizing the salt-induced activation of enzymes in organic solvents: Effects of lyophilization time and water content

The addition of simple inorganic salts to aqueous enzyme solutions prior to lyophilization results in a dramatic activation of the dried powder in org anic media relative to enzyme with no added salt. Activation of both the se rine protease subtilisin Carlsberg and lipase from Mucor javanicus resultin g from lyophilization in the presence of KCl was highly sensitive to the ly ophilization time and water content of the sample. Specifically, for a prep aration containing 98% (w/w) KCl, 1% (w/w) phosphate buffer, and 1% (w/w) e nzyme, varying the lyophilization time showed a direct correlation between water content and activity up to an optimum, beyond which the activity decr eased with increasing lyophilization time. The catalytic efficiency in hexa ne varied as much as 13-fold for subtilisin Carlsberg and 11-fold for lipas e depending on the lyophilization time. This dependence was apparently a co nsequence of including the salt, as a similar result was not observed for t he enzyme freeze-dried without KCl. In the case of subtilisin Carlsberg, th e salt-induced optimum value of k(cat)/K-m for trandesterification in hexan e was over 20,000-fold higher than that for salt-free enzyme, a substantial improvement over the previously reported enhancement of 3750-fold (Khmelni tsky, 1994). As was found previously for pure enzyme, the salt-activated en zyme exhibited greatest activity when lyophilized from a solution of pH equ al to the pH for optimal activity in water. The active-site content of the lyophilized enzyme samples also depended upon lyophilization time and inclu sion of salt, with opposite trends in this dependence observed for the solv ents hexa ne a nd tetrahydrofuran. Finally, substrate selectivity experimen ts suggested that mechanism(s) other than selective partitioning of substra te into the enzyme-salt matrix are responsible for salt-induced activation of enzymes in organic solvents. (C) 1999 John Wiley & Sons, Inc.